Method and pharmaceutical composition for treating psoriasis, squamous cell carcinoma and/or parakeratosis by inhibiting expression of squamous cell carcinoma-related antigen

ABSTRACT

In a first aspect thereof, the present invention provides a method for treatment and/or prevention of a disease selected from the group consisting of psoriasis and squamous cell carcinoma by inhibiting the expression of squamous cell carcinoma antigen (SCCA) by cells. In another aspect thereof, the present invention provides a method for screening for substances that inhibit epidermal parakeratosis, wherein the activity of a candidate substance that inhibits cysteine protease inhibitory activity possessed by squamous cell carcinoma antigen 1 (SCCA-1) is used as an indicator.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No.11/886,475, which is the US National Stage application ofPCT/JP2005/018074, filed Sep. 22, 2005, which claims priority fromJapanese application JP 2005-080566, filed Mar. 18, 2005.

TECHNICAL FIELD

The present invention provides a method and pharmaceutical compositionfor treating and/or preventing a disease selected from the groupconsisting of psoriasis and squamous cell carcinoma by inhibitingexpression of squamous cell carcinoma antigen (SCCA) by cells. Inaddition, the present invention provides a method for screeningsubstances that inhibit epidermal parakeratosis based on the activity ofcandidate substances that inhibits cysteine protease activity possessedby squamous cell carcinoma antigen 1 (SCCA-1), a substance that inhibitsepidermal parakeratosis screened by such a method, and a method forinhibiting epidermal parakeratosis that normalizes keratization ofepidermal cells by inhibiting caspase-14 inhibitory activity of SCCA-1in epidermal cells.

BACKGROUND ART

SCCA is an antigen extracted from squamous epithelial cells thatdemonstrates high concentrations in blood obtained from patientssuffering from squamous cell carcinoma of the cervix, lungs, esophagusand skin and is frequently used to diagnosis squamous cell carcinoma (H.Kato et al., Cancer, 40:1621-1628 (1977); N. Mino et al., Cancer,62:730-734 (1988)). Since SCCA levels in the blood demonstrate afavorable correlation with such factors as the progressive stage ofsquamous cell carcinoma, degree of malignancy and tumor size inparticular, it is a particularly effective marker not only for earlydetection of cancer but also for evaluating the effects of cancertreatment and diagnosing the risk of recurrence.

In addition, increased expression of SCCA is also known to be observedin the upper layer of psoriatic epidermis (Takeda A. et al., J. Invest.Dermatol. (2002) 118(1), 147-154). Psoriasis is a type of skin diseasein the form of chronic and recurrent inflammatory parakeratosischaracterized by abnormal proliferation and differentiation of epidermalcells and infiltration by inflammatory cells. Psoriasis is believed tooccur due to genetic factors in addition to various environmentalfactors (Hopso-Havu et al., British Journal of Dermatology (1983) 109,77-85).

SCCA is encoded by two genes SCCA-1 and SCCA-2 arranged in tandem onchromosome 18q21.3. The proteins SCCA-1 and SCCA-2 encoded by thesegenes both have a molecular weight of about 45,000, and although theyhave an extremely high degree of homology, since they have differentamino acid sequences at the reaction site, they are believed to havedifferent functions (Schick et al., J. Biol. Chem. (1997) 27231,1849-55). Although SCCA-1 and SCCA-2 are known to be highly expressed indiseases such as squamous cell carcinoma and psoriasis, it is unclear asto what functions they perform in diseased cells.

On the other hand, keratinocytes are known to have the function offorming a protective barrier referred to as the “cornified layer”against harmful environments as a result of terminal differentiation.The terminal differentiation process is accurately controlled by adifferentiation program, and begins with proliferative basal cells, goesthrough the stages of prickle cells, granular cells and finally endswith the differentiation into keratinocytes. Dramatic changes occur bothinside and outside the keratinocytes during the transition period fromgranular cells to keratinocytes. The keratinocytes lose their nucleusand cellular organelles, while acquiring a peripheral lipid layer, astrengthened cell membrane referred to as cornified integument, and akeratin pattern. The keratin pattern maintains a flexible and tightinternal structure. According to previous reports, keratinocytesundergoing differentiation demonstrate characteristics of apoptosis suchas DNA fragmentation and TUNEL-positive cells (Haake A. R., J. Invest.Dermatol., 101, 107-12 (1993)). Caspase-like activity has been detectedin extracts of human keratinocytes, and several types of caspases areexpressed in human keratinocytes. However, other reports have indicatedthat typical pro-apoptotic caspases such as caspase-3, caspase-6 andcaspase-7 are not activated at the time of terminal differentiation.Differentiation abnormalities frequently lead to the permanent presenceof nuclei in the cornified layer referred to as “parakeratosis”.Parakeratosis causes serious damage to the barrier function of the skin.However, it has yet to be determined as to which factors are involved inthe denucleation process, and the manner in which this process isregulated during keratinocyte differentiation.

Caspases are well-known apoptotic cell death execution factors, and arecysteine proteases preserved in the evolutionary process that cleavesubstrates after aspartic acid residues. Caspases in mammals are dividedinto three subgroups according to their structure and function, namelyinitiator caspase, effector caspase and inflammatory caspase. Effectorcaspase fulfills the important role of decomposing the inhibitor ICAD ofCAD (caspase activated DNase), resulting in the dissociation of CAD asan active nuclease (Enari M. et al., Nature, 391, 43-50 (1998)). Caspaseactivity is regulated by various molecules. In particular, there arethree groups of inhibitory proteins in direct collaboration with severalcaspases. Baculovirus anti-apoptotic protein p35 inhibits caspases-1,-3, -6, -7, -8 and -10 without acting on serine or other cysteineproteinases (Zhou Q. et al., Biochemistry, 37, 10757-65 (1998)).Baculovirus also synthesizes other anti-apoptotic proteins andinhibitors of apoptosis proteins (IAP). Homologues of IAP are also foundin mammals (Verhagen A. M. et al., Genome Biol., 2, Reviews 3009(2001)). Mammalian IAP blocks apoptosis by inhibiting caspase-14 or byantagonizing pro-apoptosis-promoting factors such as DIABLO/Smac (Wu G.,Nature, 408, 1008-12 (2000)). Cytokine response modifier A (Crm A) is agenetic product of cowpox virus that is capable of inhibiting apoptoticcaspases and inflammatory caspases (Garcia-Calvo M. et al., J. Biol.Chem., 273, 32608-13 (1998)). It is quite interesting that Crm A hasbeen suggested to belong to a superfamily of serine proteinaseinhibitors, and that some serine proteinase inhibitors, such as PI-9 andPAI-1, are able to inhibit apoptosis by interacting with caspase-1 andcaspase-3, respectively (Annand R. R. et al., Biochem. J., 342Pt3,655-65 (1999)). It will therefore be interesting to determine whether ornot terminal differentiation of keratinocytes constitutes a portion ofapoptosis phenomena, and the manner in which arbitrary regulatoryproteins are involved in this process.

Caspase-14 is the newest member of the caspase family, and is expressednearly exclusively by differentiating keratinocytes. Caspase-14 wasidentified by an EST homologous among members of the caspase family.According to findings of recent research, the caspase-14 present inkeratinocytes is a processed heterodimer that demonstrates enzymaticactivity with respect to the synthetic substrate Trp-Glu-His-Asp-AFCcorresponding to caspase-1 (Mikolajczyk J. et al., Biochemistry, 43,10560-9 (2004)). This hydrolysis activity requires protein decompositionand cleavage as well as the presence of a kosmotropic salt. Although theprimary structure of caspase-14 is extremely similar to that ofinflammatory caspases such as caspase-1, -4 and -5, the expression ofcaspase-14 limited to differentiated keratinocytes has been suggested tobe involved in keratinocyte terminal differentiation in a different mode(Lippens S. et al., Cell Death Differ., 7, 1218-24 (2001)). However, theactivation mechanism of caspase-14 along with its natural substrate orregulatory factors have yet to be elucidated.

DISCLOSURE OF THE INVENTION

As a result of conducting research for the purpose of elucidating thephysiological mechanism of skin involving SCCA, the inventors of thepresent invention surprisingly found that SCCA is an anti-apoptoticfactor having an action of inhibiting cell apoptosis.

Simply speaking, the inventors of the present invention conductedstudies of the UV defense mechanism of the skin, and clearlydemonstrated that the expression of SCCA in the spinous layer andgranular layer increased prominently due to UV irradiation of humanskin. Therefore, when a stable expression system was established byinserting human SCCA-1 and SCCA-2 genes into 3T3 cells not observed toexpress SCCA, and UV radiation-induced apoptosis was investigated incells having a stable SCCA expression system, UV radiation-inducedapoptosis was clearly demonstrated to decrease significantly in each ofthe stable SCCA expression systems.

Moreover, as a result of establishing an SCCA-1 and SCCA-2 knockdown(siSCCA) cell line by RNA interference in which siRNA was constitutivelyexpressed with a pSilencer vector in HaCat cells highly expressing SCCA,and then irradiating that cell line with UV light, the apoptosis rate inthe SCCA knockdown cells was shown to be significantly higher than acontrol cell line. On the basis of these findings, the inventors of thepresent invention concluded that SCCA is a protein that has the actionof inhibiting apoptosis.

In diseases such as psoriasis and cancer associated with abnormal cellproliferation and differentiation, cancer cells are believed to escapecell death and continue to proliferate abnormally by inhibitingapoptosis. Thus, it is clear that in cells highly expressing SCCA, SCCAinhibits cell death due to its anti-apoptotic action thereby leading toabnormal proliferation of those cells. Accordingly, it is clear that itwould be possible treat and prevent diseases such as squamous cellcarcinoma and psoriasis associated with abnormal cell proliferation andthe like if it were possible to inhibit the expression of SCCA havingapoptosis inhibitory action.

In consideration of these findings, an object of the present inventionis to provide a method and pharmaceutical composition for treating andpreventing diseases associated with abnormal proliferation of SCCAhighly expressing cells, and particularly squamous cell carcinoma andpsoriasis.

Moreover, an object of the present invention is to develop a means forinhibiting and treating epidermal parakeratosis using a completely novelapproach differing from the prior art by elucidating the mechanism ofepidermal parakeratosis. When considering that there are no effectivedrugs for skin diseases such as atopic dermatitis and psoriasisassociated with parakeratosis, the present invention is expected to havea considerable effect in the fields of dermatology and cosmetics.

In a first aspect thereof, the present invention provides a method fortreatment and/or prevention of a disease selected from the groupconsisting of psoriasis and squamous cell carcinoma by inhibiting theexpression of squamous cell carcinoma antigen (SCCA) by cells.Preferably, inhibition of the expression of SCCA by cells is carried outby RNA interference of a gene encoding SCCA. In a more preferable aspectthereof, the RNA interference uses a double-stranded RNA comprising asense oligonucleotide strand containing the oligonucleotide of SEQ IDNO. 1 or mutant thereof and an antisense oligonucleotide strandcontaining the oligonucleotide of SEQ ID NO. 2 or mutant thereof. Here,a mutant of the oligonucleotide of SEQ ID NO. 1 has a sequence thathybridizes under highly stringent conditions with nucleotides atpositions 46 to 66 of a gene encoding SCCA, while a mutant of theoligonucleotide of SEQ ID NO. 2 has a sequence that hybridizes underhighly stringent conditions with a complementary strand of nucleotidesat positions 46 to 66 of a gene encoding SCCA.

In another aspect thereof, the present invention provides apharmaceutical composition for the treatment and/or prevention of adisease selected from the group consisting of psoriasis and squamouscell carcinoma by inhibiting the expression of SCCA by cells.Preferably, the pharmaceutical composition contains a double-strandedRNA that provides a short strand RNA causing RNA interference with agene encoding SCCA. In a more preferable aspect thereof, thepharmaceutical composition contains a double-stranded RNA comprising asense oligonucleotide strand containing the oligonucleotide of SEQ IDNO. 1 or a mutant thereof, and an antisense oligonucleotide strandcontaining the oligonucleotide of SEQ ID NO. 2 or a mutant thereof.Here, a mutant of the oligonucleotide of SEQ ID NO. 1 has a sequencethat hybridizes under highly stringent conditions with nucleotides atpositions 46 to 66 of a gene encoding SCCA, while a mutant of theoligonucleotide of SEQ ID NO. 2 has a sequence that hybridizes underhighly stringent conditions with a complementary strand of nucleotidesat positions 46 to 66 of a gene encoding SCCA.

In a preferable aspect thereof, the double-stranded RNA is in a form ofbeing contained in a vector such as a pSilencer vector.

Moreover, the present invention provides a method for preparing cells inwhich expression of SCCA has been inhibited. Preferably, inhibition ofSCCA expression is carried out by RNA interference of a gene encodingSCCA. In a more preferable aspect thereof, the RNA interference uses adouble-stranded RNA comprising a sense oligonucleotide strand containingthe oligonucleotide of SEQ ID NO. 1 or mutant thereof and an antisenseoligonucleotide strand containing the oligonucleotide of SEQ ID NO. 2 ormutant thereof. Here, a mutant of the oligonucleotide of SEQ ID NO. 1has a sequence that hybridizes under highly stringent conditions withnucleotides at positions 46 to 66 of a gene encoding SCCA, while amutant of the oligonucleotide of SEQ ID NO. 2 has a sequence thathybridizes under highly stringent conditions with a complementary strandof nucleotides at positions 46 to 66 of a gene encoding SCCA. Moreover,the present invention provides cells in which expression of SCCA hasbeen inhibited by the method described above and a mammal containingsuch cells.

Accordingly, the present invention provides a method and pharmaceuticalcomposition for treating and preventing a disease selected from thegroup consisting of squamous cell carcinoma and psoriasis.

In a still other aspects thereof, the present invention includes theaspects of the invention described below.

[1] A method for screening for substances that inhibit epidermalparakeratosis, wherein the activity of a candidate substance thatinhibits cysteine protease inhibitory activity possessed by squamouscell carcinoma antigen 1 (SCCA-1) is used as an indicator.[2] The method of [1] above, comprising the following assay systems (1),(2) and (3):

(1) measuring cysteine protease activity and obtaining that measuredvalue [x];

(2) i) mixing a candidate compound with an equal amount of the cysteineprotease defined in (1) above in terms of enzyme activity followed byincubation; and

-   -   ii) measuring the cysteine protease activity of the incubated        mixture of (2)i) above under the same conditions as (1) above        and obtaining that measured value [y]; and,

(3) i) mixing the target candidate in an amount equal to that used in(2)i) with SCCA-1 followed by incubation;

-   -   ii) mixing the incubated mixture of (3)i) with an equal amount        of the cysteine protease defined in (1) above in terms of enzyme        activity, and incubating under the same conditions as (2)i)        above; and    -   iii) measuring the cysteine protease activity of the incubated        mixture of (3)ii) above under the same conditions as (1) above        and obtaining that measured value [z]; wherein,

the candidate compound is determined to have activity that inhibitscysteine protease inhibitory activity possessed by SCCA-1 and isselected as a substance that inhibits epidermal parakeratosis if itsatisfies the following condition:

{[z]/[x]×100}−{100−[y]/[x]×100}>0.

[3] The method of [2] above, wherein the condition to be satisfied is{[z]/[x]×100}−{100−[y]/[x]×100}>3.[4] The method of [3] above, wherein the condition to be satisfied is{[z]/[x]×100}−{100−[y]/[x]×100}>16.[5] The method of any of [1] to [4] above, wherein the cysteine proteaseis caspase-14.[6] The method of any of [1] to [4] above, wherein the cysteine proteaseis papain.[7] A skin composition for external use, and particularly an aestheticskin composition for external use, for inhibiting epidermalparakeratosis containing as an active ingredient thereof one or aplurality of types of herbal medicines selected from the groupconsisting of cattail extract, grape extract, tomato extract, cucumberextract, kiwi extract and jujube extract.[8] The skin composition for external use of [7] above containingcattail extract.[9] The composition of [7] or [8] above, wherein the epidermalparakeratosis is caused by psoriasis.[10] The composition of [7] or [8] above, wherein the epidermalparakeratosis is caused by atopic dermatitis.[11] A method for inhibiting epidermal parakeratosis by inhibitingcaspase-14 inhibitory activity of SCCA-1 in epidermal cells to normalizeepidermal cell keratization.[12] The method of [11] above, wherein caspase-14 inhibitory activity ofSCCA-1 in epidermal cells is inhibited by applying to the skin a skincomposition for external use containing as an active ingredient thereofone or a plurality of types of herbal medicines selected from the groupconsisting of cattail extract, grape extract, tomato extract, cucumberextract, kiwi extract and jujube extract.[13] The method of [12] above, wherein the skin composition for externaluse contains cattail extract.

Accordingly, the present invention is able to provide a means forinhibiting and treating epidermal parakeratosis using a completely novelapproach differing from the prior art.

[14] The use of one or a plurality of types of herbal medicines selectedfrom the group consisting of cattail extract, grape extract, tomatoextract, cucumber extract, kiwi extract and jujube extract as an activeingredient for producing a skin composition for external use, andparticularly an aesthetic or cosmetic skin composition for external use,for inhibiting epidermal parakeratosis.[15] The use of [14] above, wherein the herbal medicine is cattailextract.[16] The use of [14] or [15] above, wherein the epidermal parakeratosisis caused by psoriasis.[17] The use of [14] or [15] above, wherein the epidermal parakeratosisis caused by atopic dermatitis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the expression of SCCA in epidermis at exposed sites andunexposed sites.

FIG. 2 shows fluctuations in expression of SCCA in epidermis due to UVirradiation.

FIG. 3 shows the effects of UV irradiation on SCCA expression incultured human keratinocytes.

FIG. 4 shows a comparison of UV irradiation-induced apoptosis betweenSCCA highly expressing cells and SCCA non-expressing cells.

FIG. 5 shows the establishment of an SSCA knockdown cell line.

FIG. 6 shows a comparison of rates of apoptosis induced by UVirradiation between SCCA knockdown cells and control cells.

FIG. 7 shows a Western blot analysis of a skin extract. The presence ofzymogen and active caspase-14 by immunoblotting using H-99 antibody andh14D¹⁴⁶ antibody. 10 μg (Lanes 1, 2 and 4) and 1 μg (Lane 3) of wholeskin extract, skin equivalent extract and keratinocyte extract wereapplied.

FIG. 8 shows an analysis of purified caspase-14. (A) Fraction No. 25obtained from Superdex 75 chromatography following SDS polyacrylamidegel electrophoresis was transferred to a PVDF membrane and stained withCoomassie brilliant blue. Two protein bands were observed at 17 KDa and11 KDa. (B) Western blot analysis using H-99, h14D¹⁴⁶ and C20antibodies. The 17 KDa band was observed to be positive for both the H99and h14D¹⁴⁶ antibodies, while the 11 KDa band was recognized with C20antibody.

FIG. 9 shows the results of various inhibitors on purified caspase-14.Caspase-14 was incubated with peptide inhibitors of caspase (consistingof YVAD, VDVAD, DEVD, VEID, IETD, LEHD and DNLD) and class-specificinhibitors of cysteine proteinase (IAA) and serine proteinase (AEBSF).Residual enzyme activity was measured using WEHD-MCA as substrate in thepresence of 1.3 M sodium citrate and 5 mM DTT. The enzymes were testedat concentrations of 5, 2.5 and 1.25 μM, respectively. The values areshown as the average values of duplicate assays.

FIG. 10 (A) shows the cleavage activity of purified caspase-14 againstICAD. (B) shows a Western blot analysis using FL331 antibody. Cleavageproducts were observed at 33 and 27 KDa. ICAD cleavage was completelyinhibited by pre-incubating using 10 μM SCCA-1. ICAD decomposition wasonly observed in the presence of kosmotropic salt. Western blot analysisusing an antibody specific for the amino terminal demonstrated a loss ofintact ICAD molecules during extended incubation with caspase-14. WhenSCCA-1 was added to the mixture, ICAD decomposition was virtually notdetected, and there were still no effects after incubating for 16 hours(B). (C) The results are shown of investigating hydrolysis activity onsynthetic caspase substrates in the presence of kosmotropic salt.

FIG. 11 shows the localization of active caspase-14 and TUNEL-positivecells. Thin sections of normal human skin were stained with H-99antibody (A), h14D¹⁴⁶ antibody (B) and TUNEL (C). Texas-Red was used forfluorescent detection (B). FITC was used for the TUNEL, Texas-Red wasused for immunostaining, and double staining was performed for TUNEL andcaspase-14.

FIG. 12 shows the co-localization of ICAD and parakeratotic nuclei. Thinsections of normal human skin were stained with anti-ICAD antibodyFL331. Nearly all subepidermal nuclei were positive for this antibody.When ICAD on the superficial layer of keratinocytes from the skin of ADpatients was stained with antibody, positive sites of various sizes wereobserved (B). Nuclear staining with propidium iodide (PI) showingnuclear clusters was frequently observed (C). Bright field observationsof the same sites indicated overlapping scales on the surface (D).Superimposed images clearly demonstrated the presence of ICAD only atsites of parakeratosis (E). Superimposed images of nuclear staining werealso indicated in bright field observations (F).

FIG. 13 shows co-localization of SCCA-1 and parakeratotic nuclei. Hardlyany SCCA-1 was able to be detected in sections of normal skin.SCCA-1-positive sites were observed in the superficial layer of the skinof AD patients (H). Nuclear clusters were only observed at these sites(I). The bright field image is shown in (J). Superimposed imagesindicated that SCCA-1-positive sites are preferably superimposed withsites where undigested nuclei are present (K). SCCA-1 staining and adifferent superimposed image corresponding to bright field observationare also shown (L).

BEST MODE FOR CARRYING OUT THE INVENTION

Method and Pharmaceutical Composition for Treating Psoriasis and/orSquamous Cell Carcinoma

The inventors of the present invention clearly demonstrated that SCCA isa protein that has action which inhibits apoptosis. Thus, it is clearthat inhibition of the expression of SCCA would make it possible totreat and prevent diseases associated with abnormal proliferation anddifferentiation of cells expressing squamous cell carcinoma antigen.Examples of squamous cell carcinoma include squamous cell carcinoma oforgans such as the cervix, lungs, esophagus, maxilla and skin.

SCCA is a protein having a molecular weight of about 45,000 that ispresent in squamous cell carcinoma cells and psoriatic epidermis aspreviously described. The amino acid sequences of SCCA-1 and SCCA-2along with the nucleic acid sequences that encode them have beendescribed by Takeda et al., J. Invest. Dermatol., 118, 147-154 (2002)(op cit).

Although inhibition of expression of SCCA by cells can be achieved byvarious genetic technologies such as RNA interference, antisense RNA-DNAmethods, peptide and RNA-DNA aptamers, site-specific deletion,homologous recombination, dominant-negative alleles and intrabodies, RNAinterference is particularly preferable.

RNA interference is a method used to inhibit expression of a target geneby inserting into cells double-stranded RNA composed of a strandcontaining a sense oligonucleotide of about 21 to 23 base pairscomplementary to a portion of mRNA encoding a portion of the targetgene, and a strand containing an antisense oligonucleotide of about 21to 23 base pairs homologous to a portion of that mRNA. This method isbased on the interference characteristics of double-stranded RNA derivedfrom a gene-coding region, and has been demonstrated to have superiorusefulness in genetic research on nematodes (Fire et al., Nature (1998)391:806-811). It can also be used to produce function deficientphenotypes in fruit flies and mammals.

In the method of the present invention, double-stranded RNA (dsRNA) issynthesized in vitro composed of a sequence complementary to a suitabletarget region of SCCA gene, and preferably a region having a nucleotidelength of about 18 to 23 nucleotides (sense oligonucleotide), and asequence having a nucleotide length of about 18 to 23 nucleotideshomologous to that sense sequence (antisense oligonucleotide).Preferably, the double-stranded RNA is composed of a senseoligonucleotide containing a sequence complementary to the nucleotidesat positions 46 to 66 of SCCA gene (ACATGAACTT GGTGTTGGCT T: SEQ ID NO.1), and an antisense oligonucleotide containing a sequence homologous tothe sequence of nucleotides at positions 46 to 66 (AAGCCAACAC CAAGTTCATGT: SEQ ID NO. 2). Although there are no particular limitations on thetotal lengths of the sense and antisense oligonucleotides, the lengthsare, for example, about 25 to 100 nucleotides, preferably 40 to 80nucleotides and more preferably 50 to 70 nucleotides.

The sense oligonucleotide may be an oligonucleotide containing a mutantof the oligonucleotide of SEQ ID NO. 1. This mutant preferably has asequence that hybridizes under high stringent conditions withnucleotides at positions 46 to 66 of a gene encoding SCCA. In addition,the antisense oligonucleotide preferably has a sequence that hybridizesunder highly stringent conditions with a complementary strand tonucleotides at positions 46 to 66 of a gene encoding SCCA. Here,hybridization under highly stringent conditions refers to conditionsincluding a sodium concentration of about 10 to 40 mM and preferablyabout 20 mM, and a temperature of about 50 to 70° C. and preferablyabout 60 to 65° C.

The resulting double-stranded RNA may be inserted directly into targetcells or may be inserted into target cells after linking dsRNA to avector having a required element for transcription such as a promoter orterminator. Although various vectors known among persons with ordinaryskill in the art can be used for the vector, the pSilencer vector(Ambion) is preferable. As a result of inserting a vector containing thedouble-stranded RNA into cells, the sense strand of SEQ ID NO. 1 (ormutant thereof), the antisense strand of SEQ ID NO. 2 (or mutantthereof), and short hairpin RNA (shRNA) that connects the sense strandand antisense strand are formed by transcription within the cells. TheshRNA is then cleaved to a short strand RNA (short interfering RNA) ofabout 21 to 23 base pairs by intracellular nuclease and dicer, and formsan RISC complex that causes RNA interference that cleaves SCCA mRNAresulting in inhibition of the expression of SCCA by the inserted cells.

Preferably, in the present invention, a gene that inhibits expression ofSCCA, such as a gene that inhibits expression of the double-stranded RNAor other SCCA, including antisense DNA or RNA and aptamer RNA or DNA (tobe simply referred to as an “SCCA expression-inhibiting gene”), is usedas an active ingredient of a pharmaceutical composition for inhibitingexpression of SCCA, thereby preventing and/or treating psoriasis orsquamous cell carcinoma. This SCCA expression-inhibiting gene may beadministered directly by injection, or may be administered to anaffected area by a method consisting of administration of a vector orplasmid incorporating that gene.

Examples of the aforementioned vector include adenovirus vector,adeno-associated virus vector, herpes virus vector, vaccinia virusvector and retrovirus vector, and the use of these vectors enables theSCCA expression-inhibiting gene to be administered efficiently. Inaddition, a method can also be employed by which the SCCAexpression-inhibiting gene is inserted into liposomes or otherphospholipid vesicles followed by administration of the liposomes. Sinceliposomes are closed vesicles containing biodegradable material, mixingliposomes and genes causes the gene to be retained in the aqueous layerand lipid bimolecular layer within the liposomes (liposome-genecomplex). Next, culturing this complex with cells results in the genewithin the complex being taken up into the cells (lipofection). Theresulting cells may be administered by an administration methoddescribed below.

The aforementioned pharmaceutical composition can be administered in anadministration form consisting of systemic administration such asordinary intravenous or intraarterial administration, or localadministration into various tissues in which a tumor or psoriasis ispresent. Moreover, an administration form can also be used that combinesa catheter procedure, surgical procedure and the like. The dosage of thepharmaceutical composition of the present invention varies according toage, gender, symptoms, administration route, number of administrationsand drug form, and is suitably determined by a physician and the like.

When plasmid DNA is administered directly into a vein, it is difficultto express a gene since the DNA is immediately decomposed by enzymessuch as DNase in the blood. Therefore, in the case of using plasmid DNA,it is preferably to use a method by which the plasmid is injecteddirectly. Since this method allows the vector to be purified more easilyas compared with methods using vial vectors, a large amount can beprepared in a short period of time, thereby offering various advantagessuch as being produce a large amount in a short period of time and theabsence of restrictions on size of the inserted gene or concentration atthe time of injection (Verma, I. M. et al., Nature 389: 239-242, 1997).

In the case of a DNA direct injection method, the purified plasmid DNAmay be dissolved in physiological saline and the like followed byinjecting directly into muscle. As a result, the plasmid DNA is taken upinto the cells in the vicinity of the injection site, expression of theincorporated gene occurs in expression units of the eukaryote of theplasmid, and the gene product is produced. Although DNA directioninjection methods currently consist mainly of intramuscular injection,plasmid DNA can also be directly injected into, for example, tumors(Yang, P. J. et al., Gene. Ther. 3: 542-548, 1996), or into the skin(Hengge, U. R. et al., J. Clin. Invest. 97: 2911-2916, 1996; Choate, K.A. et al., Hum. Gene. Ther. 8: 1659-1665, 1997).

Intramuscularly injected plasmid DNA is injected by dissolving, forexample, 1 μg to 1 mg, and preferably 25 to 100 μg of DNA in 50 μl ofphysiological saline. This enables the maximum level of expression to beobtained after 4 to 7 days.

Confirmation of inhibition of SCCA expression can be carried out by, forexample, directly measuring the amount of SCCA in cells. Preferably,this is determined by extracting RNA from the cells and measuring theamount of RNA that encodes SCCA. Extraction of RNA and measurement ofthe amount thereof are known in the art, and quantification of RNA, forexample, can be carried out by quantitative polymerase chain reaction(PCR). In addition, measurement of SCCA can be carried out by variousmethods known method in the art by utilizing specific antibody to SCCA,examples of which include immunostaining methods using a fluorescentsubstance, pigment or enzyme, Western blotting, or immunoassay methodssuch as ELISA or RIA. The expressed amount of SCCA can also be measuredby measuring a known biological activity of SCCA. In addition,expression of SCCA can also be determined through in situ hybridizationor measurement of other biological activity.

The cells in which expression of SCCA is inhibited by the presentinvention are preferably epidermal cells, and may be granular cells orspinous cells. In addition, mammals having such cells may be humans ornon-human mammals such as mice, rats, hamsters, guinea pigs, rabbits,dogs, cats, horses, cows, sheep, pigs, goats or monkeys. The mammals andcells as claimed in the present invention can be used as model animalsor cells used in, for example, the elucidation of the UV defensemechanism of the epidermis, or research, development and screening ofdrugs preventing or inhibiting UV-induced skin damage.

Screening Method for Substances Inhibiting Parakeratosis, SubstancesScreened by this Method, and Method for Inhibiting Parakeratosis

Psoriasis is a type of skin disease in the form of a chronic, recurrentinflammatory parakeratosis characterized by abnormal proliferation anddifferentiation of epidermal cells and infiltration by inflammatorycells. Psoriasis is believed to occur due to genetic factors in additionto various environmental factors (Hopso-Havu et al., British Journal ofDermatology (1983) 109, 77-85). SCCA is encoded by two genes SCCA-1 andSCCA-2 arranged in tandem on chromosome 18q21.3. The proteins SCCA-1 andSCCA-2 encoded by these genes both have a molecular weight of about45,000, and although they have an extremely high degree of homology,since they have different amino acid sequences at the reaction site,they are believed to have different functions (Schick et al., J. Biol.Chem. (1997) 27231, 1849-55).

The screening method for substances inhibiting parakeratosis as claimedin the present invention uses as an indicator the activity of acandidate substance that inhibits cysteine protease inhibitory activitypossessed by SCCA-1. Although it is ideally most preferable to usecaspase-14 for the cysteine protease, other members of the caspasefamily, or in consideration of ease of acquisition, any other known typeof cysteine protease such as papain, cathepsins such as cathepsin B orcathepsin L, bromelain or ficin, can be used as a substitute.

In a preferable aspect thereof, the aforementioned screening method iscomposed of (1) a system for assaying the activity of cysteine protease,(2) a system for assaying the activity of cysteine protease in thepresence of a candidate substance only, and (3) a system forpre-incubating a candidate substance and SCCA-1 and assaying theactivity of cysteine protease in the presence of the incubated mixture.Assay system (2) makes it possible to determine the effects of thecandidate substance on cysteine protease. Furthermore, there are noparticular limitations on the order in which assay systems (1), (2) and(3) are carried out, and the assays may be carried out on the same dayor different days provided the assay conditions are the same.

Measurement of the enzymatic activity of cysteine protease can becarried out by a method known among persons with ordinary skill in theart using a commonly used cysteine protease substrate such asNα-benzoyl-L-arginine 4-nitroanilide hydrochloride (L-BAPNA).

In a particularly preferable aspect thereof, this screening method canbe carried out in the manner described below.

(1) System for Assaying Cysteine Protease Activity

Cysteine protease is incubated for a predetermined amount of time in asuitable assay buffer such as HEPES buffer. Next, a cysteine proteasesubstrate such as L-BAPNA is added, and after incubating for apredetermined amount of time at a predetermined temperature, color isdeveloped followed by measurement of the enzyme activity [x] of cysteineprotease.

(2) System for Assaying Cysteine Protease Activity in the Presence of aCandidate Substance Only

After incubating the aforementioned assay buffer and candidate substancefor a predetermined amount of time, cysteine protease is added followedby measurement of cysteine protease activity under the same conditionsas (1). The percentage (%) of this cysteine protease enzyme activity [y]to the aforementioned [x] is determined by {[y]/[x]×100}.

The value of {[y]/[x]×100} is an indicator of the cysteine proteaseinhibitory activity possessed by a test substance as described above, orin other words, the closer that value is to 100, the lower the cysteineprotease inhibitory activity of the test substance. In addition, thevalue obtained by subtracting the value {[y]/[x]×100} from 100, namely{100−[y]/[x]×100} is also determined. In this case, the closer thisvalue is to 0, the lower the cysteine protease inhibitory activity ofthe test substance.

(3) Measurement of Enzyme Activity in a System Containing

SCCA-1, Candidate Substance and Cysteine Protease

The aforementioned assay buffer and SCCA-1 are mixed followed byaddition of a candidate substance and incubating for a predeterminedamount of time. Cysteine protease activity [z] is then measured underthe conditions of (1) or (2). The percentage (%) of this cysteineprotease enzyme activity [z] to the aforementioned [x] is determined by{[z]/[x]×100}.

This value of {[z]/[x]×100} is an indicator of the total of the cysteineprotease inhibitory activity of SCCA-1 and the cysteine proteaseinhibitory activity of the test substance itself, or in other words, thecloser this value is to 100, the lower the total inhibitory activity.

Finally, the value obtained by subtracting the value of{100−[y]/[x]×100} from the value of {[z]/[x]×100} is determined. Thelarger this difference, the lower the cysteine protease inhibitoryactivity of SCCA-1 in the system containing SCCA-1, candidate substanceand cysteine protease, thereby suggesting remarkable suppression of thecysteine protease inhibitory activity of SCCA-1 by the candidatesubstance.

A substance screened according to the method described above is presumedto be a substance that has an inhibitory effect on the cysteine proteaseinhibitory activity of SCCA-1, and further has a high possibility ofhaving a parakeratosis inhibitory function and being useful as aparakeratosis inhibitor.

Confirmation of the parakeratosis inhibitory function of such asubstance can be easily carried out by applying to skin in whichparakeratosis is occurring, such as the skin of a model animal, and thenobserving any healing effects. Thus, a screening method as claimed inthe present invention is extremely useful as a method for primaryscreening of substances that inhibit parakeratosis among an infinitenumber of candidate substances such as herbal medicines. Examples ofconditions in which parakeratosis is observed include skin diseases suchas psoriasis, atopic dermatitis, porokeratosis, solar keratosis,seborrheic keratosis and lichen planus. Accordingly, a substanceselected according to the screening method as claimed in the presentinvention is useful in the treatment and prevention of these skindiseases.

When the inventors of the present invention studied the inhibitoryeffects of various herbal medicines on the cysteine protease inhibitoryactivity of SCCA-1, it was found that cattail extract, grape extract,tomato extract, cucumber extract, kiwi extract and jujube extractpossess such inhibitory effects. Thus, in a different aspect thereof,the present invention provides a skin composition for external use thatinhibits epidermal parakeratosis by containing as an active ingredientthereof one or a plurality of types of herbal medicines selected fromthe group consisting of cattail extract, grape extract, tomato extract,cucumber extract, kiwi extract and jujube extract.

Extracts from these plants can be obtained by drying a plant rawmaterial as necessary and then thinly slicing or crushing as necessaryfollowed by extracting with an aqueous extraction agent or organicsolvent. Examples of aqueous extraction agents that can be used includecold water, warm water or hot water at the temperature of the boilingpoint or lower, while examples of organic solvents that can be usedinclude methanol, ethanol, 1,3-butanediol and ether, and these can beused either at normal temperature or after heating.

One type or two or more types of the aforementioned extracts can berandomly selected for use in the composition for external use as claimedin the present invention. The content of these extracts is preferably0.001 to 20.0% by weight, more preferably 0.01 to 10.0% by weight, andparticularly preferably 0.1 to 5.0% by weight, based on the total weightof the composition for external use. If the extract content is less than0.001% by weight, there are cases in which the effects of the presentinvention may not be adequately demonstrated, while if the extractcontent exceeds 20.0% by weight, it may be difficult to formulate into apreparation, thereby making this undesirable.

The composition for external use of the present invention may beproduced in accordance with ordinary methods, and although it can beproduced with the aforementioned extracts alone, components ordinarilyused in cosmetics, pharmaceuticals and other skin preparations forexternal use can be suitably incorporated as necessary in addition tothe aforementioned extracts, examples of which include oils,surfactants, powders, colorants, water, moisturizers, thickeners,alcohols, various skin nutrients, antioxidants, ultraviolet absorbers,fragrances and antiseptics.

Other additives can also be suitably incorporated, examples of whichinclude metal chelating agents such as disodium edetate, trisodiumedetate, sodium citrate, sodium polyphosphate, sodium metaphosphate andgluconic acid, drugs such as caffeine, tannin, verapamil and derivativesthereof, licorice extract, glabridin, hot water extract of firethornfruit, various herbal medicines, tocopherol acetate and glycyrrhizicacid and derivatives or salts thereof, whiteners such as vitamin C,magnesium ascorbyl phosphate, ascorbic acid glucoside, arbutin and kojicacid, sugars such as glucose, fructose, mannose, sucrose and trehalose,and vitamin A such as retinol, retinoic acid, retinyl acetate andretinyl palmitate.

A composition for external use of the present invention can be used as acosmetic or over-the-counter medicine, and preferably as a cosmetic,applied to the outer skin, and can be used in a wide range of drug formssuch as an aqueous liquid form, solubilized form, emulsified form,powder form, oily liquid form, gel form, ointment form, aerosol form,water-oil bilayer form or water-oil-water trilayer form. In addition,the composition for external use of the present invention can be appliedto a wide range of drug forms such as foundation in the case of makeupcosmetics or as body soap or soap in the case of toiletry products. Itcan also be applied to a wide range of drug forms such as various typesof ointments in the case of over-the-counter medicines. Those forms thatcan be adopted by the composition for external use of the presentinvention are not limited by these drug forms or product types.

In addition, the present invention provides a method for inhibitingepidermal parakeratosis by inhibiting the caspase-14 inhibitory activityof SCCA-1 in epidermal cells in order to normalize keratization ofepidermal cells. Examples of conditions in which parakeratosis isobserved include skin diseases such as psoriasis, atopic dermatitis,porokeratosis, solar keratosis, seborrheic keratosis and lichen planusas previously described. Although the present invention is preferablycarried out by applying a skin composition for external use as claimedin the present invention to the skin, there are no particularlimitations on the application method and dosage thereof, and aresuitably determined according to the drug form of the skin compositionfor external use and the status of parakeratosis of the skin to betreated.

The following provides a more detailed explanation of the presentinvention through specific examples thereof. Furthermore, the presentinvention is not limited by these examples.

EXAMPLES

(1) Method and Pharmaceutical Composition for Treatment of Psoriasisand/or Squamous Cell Carcinoma

(1-i) Immunohistochemical Examination

Biopsied epidermis was fixed with cold acetone and then embedded inparaffin in accordance with the AMeX procedure (Sato, Y., et al., Am. J.Pathol., 125, 431-435 (1986)). Thin sections were removed of paraffinwith xylene and washed with acetone and then PBS. Next, non-specificbinding sites of the thin sections were blocked with 10% normal rabbitserum (Histofine, Tokyo, Japan).

The epidermal thin sections were respectively incubated with anti-SCCA-1monoclonal antibody (Santa Cruz Biotechnology, CA, USA) (diluted 1:500),anti-SCCA-2 monoclonal antibody (Santa Cruz Biotechnology, CA, USA)(diluted 1:500) or anti-SCCA polyclonal antibody (purified as describedin Takeda, A., et al., J. Invest. Dermatol., 118, 147-154 (2002)). Afterwashing with PBS, the thin sections were counter stained withhematoxylin and observed using the Dako Envision System (Dako Corp., CA,USA).

FIG. 1 shows the results of microscopic observations using epidermisspecimens of epidermis from unexposed sites consisting of the upper arm(human, 24 years old), buttocks (human, 46 years old) and thigh (human,75 years old) and epidermis from exposed sites consisting of the cheek(human, 20 years old, 76 years old) and eyelid (human, 82 years old),and using for the antibody anti-SCCA polyclonal antibody that binds withboth SCCA-1 and SCCA-2. It can be understood from FIG. 1 that levels ofSCCA are considerably higher in the upper layer of the epidermis ofexposed sites as compared with unexposed sites. However, increasedexpression of SCCA was not observed in the basal layer even at exposedsites.

FIG. 2 shows the results of microscopic observations of the expressionof SCCA-1 and SCCA-2 in epidermis specimens consisting of human skinsubjected to UV irradiation (Torex FL205-E-30/DMR Transluminator(Toshiba Medical Supply)) and control epidermis not subjected to UVirradiation, respectively. The antibodies used for both specimensconsisted of anti-SCCA-1 monoclonal antibody and anti-SCCA-2 monoclonalantibody. It is clear from FIG. 2 that the expression of both SCCA-1 andSCCA-2 is increased as a result of irradiating human epidermis with UVlight. In addition, this increased expression was prominent in thespinous layer and granular layer of the skin.

On the basis of these findings, it was clearly shown that expression ofSCCA-1 and SCCA-2 is increased in the epidermis, and particularly in thespinous layer and granular layer, when the epidermis is irradiated withUV light.

(1-ii) Quantitative PCR Experiment

Next, an experiment was conducted to confirm that expression of SCCA-1and SCCA-2 in the epidermis is increased by UV irradiation at the genelevel.

Human keratinocytes were cultured in keratinocyte SFM medium (Gibco,Invitrogen) in the presence of L-glutamine and epithelial cell growthfactor at 37° C. in a 5% CO₂ atmosphere with high humidity. Cells havinga confluent density of 60 to 70% were irradiated with UVB for 0 to 48hours. The cells were irradiated with UVB using the Torex FL205-E-30/DMRTransluminator (Toshiba Medical Supply) at an intensity of 50 mJ/cm².The control cells were not irradiated with UVB.

Total RNA from the cultured cells was isolated and purified using Isogen(Nippon Gene) in accordance with instructions provided. The expressionlevels of SCCA-1 and SCCA-2 were respectively determined by quantitativereal-time polymerase chain reaction (PCR). Briefly speaking, total RNAwas converted to cDNA using Superscript II (Invitrogen, Carlsbad,Calif.). That sample was then amplified by carrying out 40 cycles of2-step PCR using the ABI PRISM 7900HT Sequence Detection System (AppliedBiosystems, Foster City, Calif.). GAPDH (glyceraldehyde-3-phosphatedehydrogenase) was used for the internal standard.

The primers used were as described below.

SCCA-1: Forward primer: 5′-GTGCTATCTGGAGTCCT-3′ (SEQ ID NO. 3)Reverse primer: 5′-CTGTTGTTGCCAGCAA-3′ (SEQ ID NO. 4) Taq Man probe:5′-CATCACCTACTTCAACT-3′ (SEQ ID NO. 5) SCCA-2: Forward primer:5′-CTCTGCTTCCTCTAGGAACACAG-3′ (SEQ ID NO. 6) Reverse primer:5′-TGTTGGCGATCTTCAGCTCA-3′ (SEQ ID NO. 7) Taq Man probe:5′-AGTTCCAGATCACATCGAGTT-3′ (SEQ ID NO. 8) GAPDH: Forward primer:5′-GAAGGTGAAGGTCGGAGTC-3′ (SEQ ID NO. 9) Reverse primer:5′-GAAGATGGTGATGGGATTTC-3′ (SEQ ID NO. 10) Taq Man probe:5′-AGGCTGAGAACGGGAAGCTTGT-3′ (SEQ ID NO. 11)

A reporter dye (6-carboxyfluorescein) was coupled to the 5′-terminal ofthe Taq Man probe sequence, and a quencher dye(6-carboxytetramethylrhodamine) was incorporated on the 3′-terminalthereof.

FIG. 3 shows the results of the effects of UVB irradiation on theexpression of SCCA in cultured human keratinocytes. Expression of SCCA-1and SCCA-2 was clearly increased by UV irradiation. Thus, it is clearthat expression of SCCA-1 and SCCA-2 is epidermal cells is increased atthe gene level by UV irradiation.

Study of Role of SCCA in UV Irradiation

As has been described above, expression of SCCA-1 and SCCA-2 inepidermal cells is increased as a result of being subjected to UVirradiation. Next, a study was conducted as to the role played by SCCA-1and SCCA-2 in epidermal cells subjected to UV irradiation.

(1-iii) Establishment of SCCA-1 and SCCA-2 Highly Expressing Cells

3T3 cells (acquired from ATCC) are cells derived from mouse fetuses thatdo not express SCCA-1 or SCCA-2. A gene encoding SCCA-1 or SCCA-2 wasinserted into these cells as described below.

SCCA-1 and SCCA-2 cDNA (Takeda, A., et al., J. Invest. Dermatol., 118,147-154 (2002)) was double-digested with BamHI and KpnI. These were thensubcloned into a pTarget vector followed by insertion of 3T3 cells usingLipofectamine Plus (Gibco, Invitrogen). Briefly, 20 μg of cDNA in 675 μlof serum-free DMEM medium (Invitrogen Corp.) were mixed with 75 μl ofPlus reagent and allowed to stand for 15 minutes at 25° C. Lipofectamine(100 μl) was added to 650 μl of serum-free DMEM medium followed by theaddition thereof to the aforementioned cDNA-Plus mixture and allowing tostand for 15 minutes at 25° C. This cDNA mixture was then added to 10 mlof serum-free DMEM medium, and 3T3 cells were incubated therein for 4hours at 37° C. in a 5% CO₂ atmosphere. The medium was replaced withDMEM medium containing 10% FCS (Invitrogen Corp.) and then incubatedovernight. On the following day, G418 (Calbiochem) was added to a finalconcentration of 500 μg/ml. The G418 was maintained at thisconcentration throughout the culturing period. The medium was replacedevery 2 to 3 days. After culturing for 4 weeks, several G418-resistantcolonies were able to be isolated, and SCCA-1 and SCCA-2 expressing cellsystems were established.

Cells inserted with cDNA encoding SCCA-1 (SCCA-1-inserted cells) wereconfirmed to specifically and stably express SCCA-1, while cellsinserted with cDNA encoding SCCA-2 (SCCA-2-inserted cells) wereconfirmed to specifically and stably express SCCA-2. In addition, 3T3cells inserted with a non-specific sequence using the same procedure(control cells) did not express SCCA-1 or SCCA-2.

A study was conducted on the roles played by SCCA-1 and SCCA-2 whenepidermal cells were subjected to UV irradiation using theaforementioned SCCA-1-inserted cells, SCCA-2-inserted cells and controlcells. More specifically, a study was made of the roles of SCCA-1 andSCCA-2 with respect to UV-induced apoptosis in epidermal cells.

Each of the aforementioned cells was cultured in DMEM medium containing10% FCS at 37° C. in a 5% CO₂ atmosphere at high humidity. Cells havinga cluster density of 60 to 70% were irradiated with UVB for 0 to 48hours. The cells were irradiated with UVB using the Torex FL205-E-30/DMRTransluminator (Toshiba Medical Supply) at an intensity of 50 mJ/cm².

Apoptosis was evaluated for these cells using the FACS Coulter (EPIXXL-MCL, Beckman Coulter), and analyzed by FACS (fluorescent activatedcell sorting) using double staining with Annexin V-FITC and propidiumiodine (PI) (Annexin V-FITC Kit, Immunotech) for the indicator.

Those results are shown in FIG. 4. As is clear from FIG. 4, apoptosisattributable to UV irradiation was observed to decrease significantlyfor both the SCCA-1 and SCCA-2-inserted cells. Thus, SCCA-1 and SCCA-2were presumed to be able to inhibit apoptosis induced by UV light.

In order to confirm this finding, the inventors of the present inventionestablished SCCA-1 and SCCA-2 knockdown cell lines by RNA interferenceto further examine the roles of SCCA-1 and SCCA-2 in epidermal cellssubjected to UV irradiation.

(1-iv) Establishment of SCCA Knockdown Cells

HaCat cells (H. Hans, et al., Experimental Cell Research, 239, 399-410(1998)) are human keratinocytes that highly express SCCA. SCCA-1 andSCCA-2 knockdown cell lines were established by constitutivelyexpressing siRNA (short interference RNA) with a pSilencer vector(Ambion) in accordance with the procedure for RNA interference.

The siRNA was constructed using pSilencer vector according to theinstructions provided. More specifically, a double-strandedoligonucleotide consisting of a 65 mer sense oligonucleotide (SEQ ID NO.12) containing a 21 mer oligonucleotide (ACATGAACTT GGTGTTGGCT T: SEQ IDNO. 1) complementary to nucleotides at positions 46 to 66 of a geneencoding SCCA, and a 65 mer antisense oligonucleotide (SEQ ID NO. 13)containing a 21 mer oligonucleotide (AAGCCACAAC CAAGTTCATG T: SEQ ID NO.2) homologous with nucleotides at positions 46 to 66, was cloned to theHindIII site and BamHI site of pSilencer vector. Transfection to HaCatcells was carried out using Lipofectamine 2000 (Invitrogen) inaccordance with the instructions provided. Control cells were preparedby using a double-stranded oligonucleotide consisting of twooligonucleotides neither significantly homologous or complementary tomammalian gene sequences. A stable cell system was acquired by selectingtransfected cells in hygromycin B selective medium after culturing for 4to 6 weeks. The procedure described above was used to confirm whetherexpression of SCCA was inhibited, and expression of SCCA-1 and SCCA-2was measured by real-time PCR.

Sense oligonucleotide (SEQ ID NO. 12)GATCCCGGCCAACACCAAGTTCATGTTTCAAGAGA ACATGAACTTGG TGTTGGCTTTTTTGGAAA(underline indicates homologous region) Antisense oligonucleotide(SEQ ID NO. 13) AGCTTTTCCAAAA AAGCCAACACCAAGTTCATGTTCTCTTGAAACATGAACTTGGTGTTGGCCGG

(underline indicates complementary region)

Those results are shown in FIG. 5. Expression of SCCA-1 and SCCA-2 wasconfirmed to be inhibited (knocked down) by 90% or more in cellsinserted with siRNA as described above in comparison with the controlcells.

A study was then made on the roles of SCCA-1 and SCCA-2 with respect toUV-induced apoptosis in epidermal cells using these knockdown cells andcontrol cells.

Each of the cells was cultured in keratinocyte SFM medium (Gibco,Invitrogen) in the presence of L-glutamine and epithelial cell growthfactor at 37° C. in a 5% CO₂ atmosphere with high humidity. Cells havinga confluent density of 60 to 70% were irradiated with UVB. The cellswere irradiated with UVB using the Torex FL205-E-30/DMR Transluminator(Toshiba Medical Supply) at an intensity of 50 mJ/cm².

Apoptosis was evaluated for these cells using the FACS Coulter, andanalyzed by FACS (fluorescent activated cell sorting) using doublestaining with Annexin V-FITC and propidium iodine (PI) (Annexin V-FITCKit, Immunotech) for the indicator.

Those results are shown in FIG. 6. As a result of irradiating knockdowncells with UV light, in contrast to apoptosis occurring in 38% of thecontrol cells, apoptosis was induced in roughly 80% of the knockdowncells. Thus, SCCA was determined to significant inhibit apoptosis ofepidermal cells induced by UV irradiation. Accordingly, SCCA was clearlydetermined to be responsible for the UV defense mechanism of epidermalcells, while also being a protein having an action that inhibitsapoptosis.

(2) Method for Screening for Substances Inhibiting Parakeratosis,Substances Screened by this Method and Method for InhibitingParakeratosis

(2-i) Materials and Methods

Materials

Ac-WEHD-MCA, Ac-YVAD-MCA, Ac-VDVAD-MCA, Ac-DEVD-MCA, Ac-VEID-MCA,Ac-IETD-MCA and Ac-LEHD-MCA were purchased from Peptide Institute, Inc.(Osaka, Japan).

Benzyloxycarbonyl (Z)-YVAD-FMK, Z-VDVSD-FMK, Z-DEVD-FMK, Z-VEID-FMK,Z-IETD-FMK, Z-LEHD-FMK and Z-VAD-FMK were purchased from BioVision(Mountain View, Calif.). Recombinant caspases-1 to 10 were obtained fromBIOMOL Research Labs, Inc. (Plymouth Meeting, Pa.).

H-99 antibody (Santa Cruz Biotechnology, Inc.) was used to detect thepro-forms and large subunits of caspase-14. H-99 antibody is an antibodythat occurs in response to peptides corresponding to amino acids 24 to122 of human caspase-14, and therefore reacts with proenzymes ofcaspase-14 and their processed forms, namely their large subunits.

C-20 antibody (Santa Cruz Biotechnology) was used to detect smallsubunits of caspase-14. Cleavage site-specific antibody (h14D¹⁴⁶) wasprepared immunizing a rabbit with a synthetic pentapeptide TVGGDequivalent to the presumed processing site of human caspase-14.

(2-ii) Measurement of WEHD-MCA Hydrolysis Activity

Caspase-14 activity was measured using Ac-WEHD-MCA as substrate byadding some degree of variation to the method described in Mikolajczyk,J. et al., Biochemistry, 43, 10560-9 (2004). Briefly, the assay mixturewas prepared from 45 μl of 0.1 M HEPES buffer (pH 7.5), 0.06 M NaCl,0.01% CHAPS, 5 mM DTT, 1.3 M sodium citrate and 10 μM WEHD-MCA (allconcentrations indicate the final concentrations). An enzyme sample (5μl) was added to this mixture followed by incubating for 10 to 30minutes. The reaction was stopped with 150 μl of 0.1 M monochloroaceticacid, and measurements were performed at an excitation wavelength of 355nm and emission wavelength of 460 nm using Fluoroskan Ascent FL (ThermoElectron Co., Wolsam, Mass.). In the case of the inhibitor assay,caspase-14 and peptide inhibitor were incubated in the assay buffer for15 minutes, and the assay was started with the addition of 5 μl of 100μM WEHD-MCA.

(2-iii) Purification of Caspase-14

Human cornified cells (approx. 14 g) scraped from the heels of healthyindividuals were extracted with 0.1 M Tris-HCl (pH 8.0) containing 0.14M NaCl using a glass homogenizer. A supernatant was obtained aftercentrifuging at 15,000 g for 60 minutes. After concentrating with AmiconUltra (Millipore, Mass.) and desalting with the Fast Desalting ColumnHR10/10 (Amersham Biosciences), the crude product was applied to aHiPrep 16/10 QXL column. The column was washed with 20 mM Tris-HCl (pH8.0) and eluted at a linear NaCl gradient of 0 to 1 M. The fractionswere traced by Western blotting using anti-caspase-14 antibody (H-99)(Santa Cruz Biotechnology, CA) and h14D¹⁴⁶ antibody. In addition,hydrolysis activity with respect to Ac-Tyr-Glu-His-Asp-methyl-coumarinamide (WEHD-MCA) (Peptide Institute, Inc., Osaka, Japan) was measuredfor each fraction. Positive fractions were placed on a Mono Q columnequilibrated with the same buffer and eluted a maximum NaCl gradient of1 M. The caspase-14 fraction was further separated by Mono S cationicexchange chromatography. The column was equilibrated with 20 mM acetatebuffer (pH 4.5) and eluted at an NaCl gradient of 0 to 1 M. Positivefractions were concentrated and placed on a chromatofocusing Mono Pcolumn equilibrated with 25 mM ethanolamine (pH 8.3). The column waseluted while forming a pH gradient from 8 to 5 using 46 ml of Polybuffer(pH 5.0). The caspase-14 was finally purified using Superdex 75 gelchromatography. The protein concentration was determined with the BioRadProtein Assay Kit (BioRad Lab, Hercules, Calif.).

(2-iv) Preparation of Recombinant Caspase-14 and SCCA-1

cDNA encoding caspase-14 was isolated and amplified from keratinocytecDNA by PCR using a forward primer in the form ofAAGGATCCAATCCGCGGTCTTTGGAAGAGGAG (SEQ ID NO. 14) and a reverse primer inthe form of TTTCTGCAGGTTGCAGATACAGCCGTTTCCGGAGGGTGC (SEQ ID NO. 15). ThePCR product was cloned in a pQE-100 DoubleTag vector (Qiagen, Valencia,Calif.) and expressed in E. coli JM109.

SSCA1 cDNA was separated from a cDNA library (Takeda, A., et al., J.Invest. Dermatol., 118, 147-154 (2002)) and cloned in pQE30 vector(Qiagen). Recombinant protein was purified with Ni-NTA Agarose (Qiagen)and Mono Q chromatography.

(2-v) Immunohistochemistry

Human scalp samples were obtained by plastic surgery after acquiringpatient consent. The tissue was fixed with 4% paraformaldehyde (PFA) inphosphate buffer solution (pH 7.4) and then embedded in paraffin. Thinsections were then prepared followed by incubation with suitableantibody overnight at 4° C. Peroxidase-coupled goat anti-rabbit IgG(Nichirei Corp.) was used as secondary antibody and reacted with a colordeveloping reagent in the form of DAB.

In the case of double staining detection of TUNEL-positive cells andactive caspase, Texas Red (registered trademark) dye-coupled anti-rabbitIgG (Rona) was used as secondary antibody. The TUNEL reaction wascarried out using a fluorescein in situ cell death detection kit (RocheDiagnostics) in accordance with the instructions provided by themanufacturer.

Antibodies ICAD IgG (FL331, Santa Cruz Biotechnology) and DFF45/ICADAb-2 (NeoMarkers, Fremont, Calif.) were used in the case of ICAD Westernblotting and immunohistochemical analysis.

Clustered parakeratosis has been reported to be frequently observed inthe skin of active atopic dermatitis (AD) patients (Sakurai, K., et al.,J. Dermatol. Sci., 30, 37-42 (2002); Piloto Valdes, L., et al.,Allergol. Immunopathol. (Madr), 18, 321-4 (1990)). In this experiment,the localization of ICAD and SCCA-1 in parakeratotic skin wasinvestigated using a non-invasive method. A sample of the superficialcornified layer was collected from the skin of an AD patient and healthyvolunteer, and adhered to slide glasses using the medical adhesive, AronAlpha A (Sankyo Co., Tokyo). After fixing with 3% formaldehyde, thesamples were permeated with 0.1% Triton X-100 followed by immunostainingwith anti-ICAD or anti-SCCA-1 antibody overnight at 4° C. Alexa Fluor400-coupled anti-rabbit antibody (ICAD) or anti-mouse IgG (SCCA-1)antibody were respectively used as secondary antibody at roomtemperature for 1 hour. The samples were immersed for 5 minutes in 0.1%pyridium iodide to visualize the nuclei and then washed three times withPBS. The Leica DMLA microscope was used for fluorescent microscopy.

(2-vi) Western Blot Analysis

Protein was separated by SDS-polyacrylamide gel electrophoresis at aconcentration gradient of 5 to 20%. Following electrophoresis, theprotein was transferred to a polyvinylidene difluoride membrane(Immobilon-P, Millipore, Bedford, Mass.) and then incubated withanti-caspase-14 antibody containing H-99, h14D¹⁴⁶ or C20. Immunoreactiveprotein was visualized by chemiluminescence with the ECL-Plus (Amersham)using peroxidase-labeled anti-rabbit IgG (Sigma) or anti-goat IgG assecondary antibody.

(2-vii) Results

Keratinocyte Caspase-14 Processed with Asp¹⁴⁶

According to Western blot analysis, only a 17 KDa band was detected inkeratinocyte extract for H-99 antibody (FIG. 8). This result does notagree with the result for extracts derived from whole skin orskin-equivalent models containing an unprocessed 30 KDa form. This 17KDa band was also recognized with h14D¹⁴⁶ antibody (FIG. 8B), and ispresumed to be a large subunit of active caspase-14 (p17). This suggeststhat maturation of caspase-14 is achieved by cleavage at Asp¹⁴⁶ duringthe final stage of terminal differentiation. Moreover, the 30 KDa bandwas also recognized by H-99 and h14D¹⁴⁶ antibody in a skin-equivalentmodel, suggesting cleavage at Asp¹⁴⁶.

(2-viii) Preparation of Caspase-14 from Keratinocyte Extract

The majority of the caspase-14 in keratinized cells is in a processedform, and for this reason, since it presumed to be present in an activeform (Eckhart, L., et al., J. Invest. Dermatol., 115, 1148-51 (2000);Lippens, S., et al., Cell Death Differ., 7, 1218-24 (2000); Mikolajczyk,J., et al., Biochemistry, 43, 10560-9 (2004)), human keratinocytes arethought to be a superior source of purified caspase-14. However, humankeratinocytes are also known to contain caspase-1-like enzyme(Takahashi, T., et al., Invest. Dermatol., 111, 367-72 (1998)).Substrates of caspase-1 such as WEHD-substrate can be hydrolyzed by bothcaspase-1 and caspase-14. The inventors of the present invention firsttested hydrolysis of WEHD-MCA by caspase-1 with 1.3 M sodium citrate andin the presence and absence of 5 mM dithiothreitol. Despite WEHD-MCAbeing a superior substrate of caspase-1 in standard caspase assaybuffer, caspase-1 was confirmed to be unable to hydrolyze this substratein the presence of kosmotropic ion (data not shown). Thus, each fractionwas evaluated by three methods consisting of hydrolysis activity onWEHD-MCA, reactivity with H-99 and h14D¹⁴⁶ antibody. Table 1 shows theresults of continuous chromatography. Following the initial anionicexchange chromatography using a HiPrep Q column, the yield demonstratedan increase of 170%, while specific activity increased roughly ten-fold.This increase was probably attributable to the dissociation ofcaspase-14 from an intrinsic inhibitor. According to Western blotanalysis, fractions nos. 16 to 20 were shown to contain 17 KDa bandspositive for H-99 and h14D¹⁴⁶. These fractions were also observed todemonstrate WEHD-MCA hydrolysis activity. In the subsequent Mono Qanionic exchange chromatography, fractions Nos. 25 to 29 contained aprocessed form of caspase-14 in accordance with an assessment made basedon the presence of 17 KDa bands positive for H-99 and h14D¹⁴⁶. Onlythese fractions were observed to demonstrate WEHD-MCA hydrolysisactivity. Mono S cationic chromatography and Mono P chromatofocusingwere effective for removing the major contaminant proteins, and specificactivity increased 3.5-fold and 7-fold, respectively. Here again, onlythe H-99 and hl4D¹⁴⁶-positive fractions demonstrated WEHD-MCA hydrolysisactivity. In the final stage using Superdex 75 chromatography, a peakhaving a molecular weight of 30 KDa was separated, and this peakcoincided with the peak for WEHD-MCA hydrolysis activity. InSDS-polyacrylamide gel electrophoresis, this preparation was shown tocontain 17 KDa and 11 KDa fragments. The former was positive for bothH-99 antibody and h14D¹⁴⁶ antibody, while the latter was recognized withC20 antibody. This suggests that human caspase-14 is purified in theform of a heterodimer composed of a large subunit (17 KDa) and a smallsubunit (11 KDa). In addition, Superdex 75 chromatography indicatedthat, differing from other caspases, caspase-14 in human keratinocytesis present in the form of a monomer in the manner of a granzymeB-activated form. Table 1 provides a summary of the purification ratesof caspase-14. 11.8 μg of purified protein were obtained starting fromabout 100 mg of soluble protein extract. Specific activity increased764-fold and the yield was 9.1%.

TABLE 1 Summary of Purification of Caspase-14 Protein Total proteinEnzyme Specific Total concentration Volume weight activity activityactivity Yield μg/ml ml μg AFU (mU) (mU/mg protein) U (%) CC TBS Ext.312.0 320.00 99840.0 3.67 11.8 587.34 100.0 HiPrep Q 1185.0 15.0017775.0 133.59 112.7 1001.92 170.6 Mono Q 582.0 8.00 4656.0 150.30 258.2601.19 102.4 Mono S 61.3 8.00 490.4 56.94 928.8 227.75 38.8 Mono P 50.51.00 50.5 338.31 6699.2 169.15 28.8 Superdex 75 11.8 1.00 11.8 106.529015.1 53.26 9.1(2-viv) Enzyme Characteristics of Purified Caspase-14

The enzyme characteristics of purified caspase-14 were investigated(FIGS. 9 and 10). Caspase-14 had sensitivity to various caspaseinhibitors such as YVAD-FMK (caspase-1 inhibitor), VDVAD-FMK (caspase-2inhibitor), DEVD-FMK (caspase-3 inhibitor), IETD-FMK (caspase-8inhibitor), LEHD-FMK (caspase-9 inhibitor) and VAD-FMK (pan-caspaseinhibitor) (FIG. 9). Furthermore, VEID-FMK had hardly any effect.YVAD-FMK in particular demonstrated extremely potent inhibitory effectson caspase-14 activity. This is probably due to the structuralsimilarity between caspase-1 and caspase-14. The pan-caspase inhibitor,VAD-FMK, inhibited caspase activity to about the same degree as VAD-FMK.The cysteine protease class-specific inhibitor, iodoacetic acid (IAA),or serine protease class-specific inhibitor,4-(2-aminoethyl)benzenesulfonyl fluoride (ABBSF), did not exhibitsignificant inhibitory effects at the test concentrations used in thisstudy.

(2-x) Effects of Purified Caspase-14 on ICAD Decomposition

The inventors of the present invention tested the effects of caspase-14on ICAD during the course of searching for natural substrates ofcaspase-14. This is because disappearance of the nucleus is an extremelyimportant event for terminal differentiation. According to an assessmentmade by Western blot analysis using anti-ICAD IgG when recombinant ICADprotein and purified caspase-14 were incubated in an ordinary caspaseassay buffer, purified caspase-14 did not demonstrate any hydrolysisactivity on ICAD (FIG. 10A). However, since the amount of intact ICADprotein decreases and two major decomposition products increase in thepresence of kosmotropic salt, purified caspase-14 demonstrated limiteddecomposing action on ICAD.

(2-xi) Inhibition of Caspase-14 by SCCA-1

Although SCCA-1 is a member of the serpin superfamily, it inhibitscysteine proteases such as papain and cathepsin L (Takeda, A., et al.,Biol. Chem., 383, 1231-6 (2002)). This indicates that SCCA-1 is a uniqueclass inhibitor in the manner of Crm-A³². Thus, the inventors of thepresent invention conducted a test as to whether or not SCCA-1 is ableto inhibit caspase members. Kosmotropic conditions were used in the caseof caspase-14. None of the caspase members (1 to 10) were affected withrespect to enzyme activity when recombinant caspase was incubated withSCCA-1 (FIG. 10C). SCCA-1 inhibited decomposition of ICAD by caspase-14.Recovery of enzyme activity was not observed even if the incubation timeincreased. This suggests that strong binding between caspase-14 andSCCA-1 (FIG. 10B).

(2-xii) Localization of Active Caspase-14 and TUNEL-Positive Cells

In order to investigate whether caspase-14 is involved in thedenucleation process, the inventors of the present invention carried outcaspase-14 and TUNEL double staining. As shown in FIG. 11A, caspase-14containing pro-forms and active forms was localized in cells rangingfrom prickle cells to keratinocytes in normal human epidermis. Thisagrees with previous findings (Lippens, et al. (2000), op cit). Activecaspase-14 detected with h14D¹⁴⁶ antibody was limited to somekeratinocytes and granular cells (FIG. 11B). Nearly all keratinocyteswere unevenly stained. Although TUNEL-positive cells were observedimmediately beneath the cornified layer, the localization of themajority of these positive cells was extremely limited (FIG. 11C). It isinteresting to note that TUNEL-positive cells were nearly alwayslocalized with h14D¹⁴⁶-positive cells. This suggests that DNAfragmentation is occurring in these cells, and that active caspase-14 isinvolved in this process.

(2-xiii) Co-Localization of ICAD and SCCA-1 in Parakeratotic Nuclei

The use of FL331 antibody reveals that ICAD is primarily localized inthe nuclei of basal cells and basal epithelial cells in longitudinalcross-sections of normal human skin. The cytoplasm was weakly positivein cells ranging from basal cells to granular cells. In the cornifiedlayer, immunoreactivity to ICAD decreased considerably. Substantiallythe same results were obtained even when using the N-terminal peptideantibody, DFF45/ICAD Ab-2 (data not shown). In the case of staining thesuperficial cornified layer of AD patients with anti-ICAD antibody(FL-331), various sizes of cluster regions were positive for thisantibody (FIG. 12B). Nuclear staining by PI indicated that parakeratoticnuclei are consistently found in these porphyritic islands (FIG. 12C).Bright field observations of the superficial cornified layer revealed acoarse surface with numerous surface irregularities (FIG. 12D).Superimposed images indicated that parakeratotic sites coincided withICAD-positive sites (FIGS. 12E and 12F). These results suggest thatdecomposition of ICAD is required for elimination of nuclei duringterminal differentiation.

Extremely low levels of SCCA-1 were detected in the granular layer inthe case of normal human skin. Strong immunostaining with a prominentporphyritic distribution of positive sites was observed in thesuperficial cornified layer of active AD as well. (FIG. 13A). Similarly,SCCA-1-positive regions coincided with the PI-positive nucleus layer,namely parakeratotic sites (FIGS. 13J to 13L). In summary, these resultssuggest that the ICAD/CAD system plays an important role in thedenucleation process, and that SCCA-1 is involved in this reaction as asuppressor.

Discussion

The majority of caspase-14 is expressed in the epidermis, while hardlybeing expressed at all in other tissues (Van de Craen, M., et al., CellDeath Differ., 5, 836-46 (1998)). According to previous reports,terminal differentiation of keratinocytes is linked with the processingof caspase-14, and this suggests the activation of caspase familyproteinases (Lippens, S. et al., Cell Death Differ., 7, 1218-24 (2000);Eckhart, L., Biochem. Biophys. Res. Commun., 277, 655-9 (2000); Hu, S.,J. Biol. Chem., 273, 29648-53 (1988)). More recently, Mikolajaczyk etal. (2004, op cit) demonstrated that granzyme B-cleaving caspase-14 isenzymatically active in the presence of kosmotropic salt. In this study,the inventors of the present invention attempted to purify caspase-14from completely differentiated keratinocytes to investigate whether ornot human caspase-14 is active at the final stage of keratinocytedifferentiation. The inventors of the present invention used three typesof antibodies that recognize a large subunit, pro-form (H-99, having apresumed caspase cleaving site at Asp¹⁴⁶ (h14D¹⁴⁶)) and small subunit(C20), respectively. The final preparation was composed of two bands,namely a 17 KDa band recognized by h14D¹⁴⁶ antibody, and an 11 KDa bandrecognized by C20 antibody. These protein bands are the large and smallsubunits of active caspase-14. During the purification process, anH99-positive 17 KDa band was recognized together with h14D¹⁴⁶ antibody.This suggests that the carboxyl terminal of the large subunit ends withAsp¹⁴⁶. The amino terminal region of the small subunit was identified asLys¹⁵³-Asp-Ser-Pro-Gln, and this suggests that processing occurs betweenIle¹⁵² and Lys¹⁵³. This site is a specific cleaving site for members ofthe caspase family. This agrees with the finding of Chien, et al.(Biochem. Biophys. Res. Commun., 2002, Aug. 30: 296(4): 911-7) thatcaspase-14 immunoprecipitated from foreskin extract demonstratescleavage at the same site. Thus, it was concluded that human caspase-14is homogeneously purified in the form of a highly active heterodimer.Processing at the two sites of Asp¹⁴⁶ and Ile¹⁵² and removal of sixresidues within linker regions Xxx¹⁴⁷ and Ile¹⁵² were suggested to beinvolved in the maturation of caspase-14. The presence of two differentcleavage sites (one being acidic and the other being hydrophobic) alsosuggests that activation of caspase-14 is carried out in multiple stepsby numerous enzymes.

The enzymatic characteristics of purified caspase-14 were extremelyunique. Purified caspase-14 exhibited a comparatively broad range ofinhibitor sensitivity to known caspase inhibitors. In particular, thecaspase-1 inhibitor, YVAD-FMK, exhibited the most potent inhibitoryaction. YVAD-FMK also demonstrated higher activity than WEHD-MCA andanother caspase-1 substrate in the form of YCAD-MCA. This suggests thatcaspase-1 and caspase-14 are in an intimate relationship. However, theinventors of the present invention clearly showed that caspase-14 hasconsiderably different characteristics. The inventors of the presentinvention determined for the first time that SCCA-1 is an intrinsicinhibitor against caspase-14. The most unique characteristic of SCCA-1is its remarkable specificity for caspase-14. Other members of caspase-1to -10 are not affected by SCCA-1. SCCA-1 did not inhibit caspase-3activity using the synthetic substrate DEVD-MCA or the natural substrateICAD. Crm A belongs to the serpin superfamily and is known to be able toinhibit a large number of caspases including caspase-1 and caspase-8(Gagliardini, V., et al., Science 263, 826-(1994)). XIAP is known toinhibit caspase-3, -7 and -9 (Srinivasula, S. M., et al., Nature 410,112-6 (2001)). Since the anti-apoptotic protein p35 inhibits caspase-1,-3, -6, -7, -8 and -10, it is considered to have a broader spectrum. Thefact that all of these inhibitory proteins consisting of Crm A, IAP andp35 are able to inhibit a portion of several initiator and effectorcaspases suggests that these molecules are involved in execution of thetypical apoptosis pathway. On the other hand, the results obtained bythe inventors of the present invention also strongly suggest that,although SCCA-1 is not a key factor in ordinary apoptotic phenomena, itis an important regulator in the denucleation process mediated bycaspase-14.

The molecular mechanism of this process has yet to be elucidated. Theinventors of the present invention clearly demonstrated that humancaspase-14 is able to decompose ICAD in the presence of cosmotropicsalt. ICAD (also referred to as DNA fragmentation factor DFF45) is aninhibitor of a magnesium-dependent endonuclease referred to ascaspase-activated DNase (CAD) (or DFF40). The ICAD/CAD system plays animportant role in the decomposition of chromosomal DNA during apoptoticcell death. ICAD bound to CAD is present in the form of an inactivecomplex. Caspase-3 exhibits limited protein decomposition against ICAD,cleaving at the two sites of Asp¹¹⁷ and Asp²²⁴. This cleavage activatesCAD and initiates DNA decomposition (Nagata, S., Exp. Cell Res., 256,12-8 (2000)). Although caspase-3 is not necessarily required forcleavage of a large number of cellular proteins during apoptosis, it isessential for cleaving ICAD (Tang, D., et al., J. Biol. Chem., 273,28549-52 (1998)). This indicates that caspase-3 is extremely importantfor DNA fragmentation, while other effector caspases such as caspase-6and caspase-7, are not important. Interestingly enough, caspase-14formed fragments resembling 12 KDa and 35 KDa from ICAD. Sequenceanalysis indicated identical cleavage sites. This suggests thatcaspase-14 can be a perfect substitute for caspase-3. Althoughcaspase-14 is able to decompose ICAD, it is clearly different frompro-apoptotic caspase-3 for the reasons indicated below. Firstly,excessive expression of caspase-14 does not induce apoptotic cell death(Van de Craen, Cell Death Differ., 5, 838-46 (1998)). Thischaracteristic is in contrast with that of caspase-3. Secondly,caspase-14 is not activated by various apoptotic stimuli (Lippens, etal. (2000), op cit). Initiator caspase and other caspase members wereunable to process pro-caspase-14. This finding agrees with previousfindings (Lippens, et al. (2000), op cit). Thirdly, synthesis ofcaspase-14 is limited to differentiating keratinocytes in adult tissue(Eckhart, L., Biochem. Biophys. Res. Commun., 277, 655-9 (2000)). Inaddition, the ability of caspase-14 with respect to ICAD cleavage isregulated by a mechanism that is considerably different from caspase-3.Decomposition of ICAD by caspase-14 requires an abnormally highconcentration of cosmotropic ion. Other caspases demonstrated hardlyactivity at this ion concentration. Overall, since the activation ofcaspase-14 is regulated by a keratinocyte differentiation program, it isconsidered to have a unique position among members of the caspasefamily.

Involvement of the ICAD/CAD system in terminal differentiation ofkeratinocytes is further supported by in vivo experiments.Immunohistochemical research has shown that ICAD is present in thenuclei of basal keratinocytes to spinous keratinocytes, is not presentin granular cells, and that the disappearance of nuclei occurs at thetime of terminal differentiation. Strong staining of ICAD has beendemonstrated at porphyritic sites in the superficial epidermis of ADpatients. These areas have slightly coarse surfaces and lowtranslucency. Groups of PI-positive, undigested nuclei were presentlocally at these areas. Other areas were not stained with anti-ICADantibody. In addition, although tape stripping tests performed on theskin of AD patients revealed the presence of intact ICAD protein, thiswas not detected in extracts from normal subjects. These results suggestthat ICAD is involved in the denucleation process at the time ofterminal differentiation.

There is hardly any expression of SCCA-1 in normal skin. On the otherhand, SCCA-1 is strongly expressed in psoriatic skin, mucous membranesand the esophagus (Takeda, A., et al., J. Invest. Dermatol., 118, 147-54(2002)). Interestingly enough, these tissues are accompanied byparakeratosis. The inventors of the present invention clearlydemonstrated strong staining of SCCA-1 is also found at sites ofparakeratosis. Moreover, SCCA-1 and ICAD were always present locally atthe same sites as sites where groups of nuclei were present. Since therewere no other areas of the skin surface where SCCA-1 or ICAD isnegative, the co-localization of these molecules at parakeratotic sitessuggests that these molecules are involved in inhibition of thedenucleation process. Nuclei were reported not to disappear in askin-equivalent model due to the pan-caspase inhibitor VAD-FMK (Weil, etal., 1999). The finding of the inventors of the present invention thatVAD-FMK is one of the most potent caspase-14 inhibitors enhances thepossibility that caspase-14 is probably a candidate for this reaction.Caspase-14 is down-regulated at parakeratotic sites of psoriatic skin,and is not present in epidermis of the oral cavity. In epidermis of theoral cavity, the disappearance of nuclei is either lost in some form, oris simply not carried out (Lippens, et al. (2000) op cit). Interestinglyenough, SCCA-1 is up-regulated in these tissues. Most likely theabnormal expression of these molecules causes incompletedifferentiation, including the permanent presence of nuclei.

The mechanism by which caspase-14 is activated in the skin is not fullyunderstood. Activation is only observed in skin or a skin-equivalentmodel, and is not observed in cultured cells (Eckhart, L., et al. (2000)op cit). The inventors of the present invention actually tested thisunder various conditions. These conditions included addition of serum,prolongation of the duration of culturing to day 14 in the presence orabsence of calcium after the cells became dense, treatment by calciumionophore A23187, and exposure to air for 30 minutes, which is adequatefor up-regulating numerous differentiation markers. Although thesedifferentiation stimuli induced prominent expression of caspase-14 mRNA,they were not effective for inducing caspase-14 activity (data notshown). The activation process is strictly controlled and is stronglyinhibited in monolayer cultures. Layering and exposure to air arethought to be required for activation. It is clear that control ofactivation of caspase-14 is not mediated by an apoptosis program, butrather mediated by a differentiation program. During the terminaldifferentiation process, numerous proteinases are activated, includingserine, cysteine and aspartic acid proteinases. Trypsin-like andchymotrypsin-like serum proteinases are suggested to fulfill the role ofexfoliating the outermost keratinocytes. Some cysteine proteinases, suchas cathepsin B and L, are up-regulated in differentiated keratinocytes.Cathepsin D and aspartic acid proteinase are also suggested to fulfillthe role of exfoliating keratinocytes. These enzymes may also beinvolved in other differentiation mechanisms such as the activation ofcaspase-14.

In summary of the above, the inventors of the present invention purifiedcaspase-14 from human keratinocyte extract. Although caspase-14 inducesdisappearance of nuclei and their disappearance resembles apoptosis, itis strongly suggested to be a distinguishable change that occursmediated by decomposition of ICAD in the final stage of keratinocytedifferentiation. Although this process shares several apoptotic factors,it does not involve cell death leading to elimination of damaged cells,but rather is a constitutional process for the purpose of completingoverall structure to demonstrate the primary role in the form of abarrier function. Abnormal expression of caspase-14 or SCCA-1 has adirect effect on the differentiation program and as a result, leads tothe occurrence of parakeratosis and a breakdown of the barrier function.

(2-xiv) Screening Method

Screening for substances that inhibit parakeratosis was carried out inthe manner described below. The herbal medicines indicated below weretested.

-   -   Cattail extract (Ichimaru Pharcos Co., Ltd.)    -   Grape extract (Ichimaru Pharcos Co., Ltd.)    -   Tomato extract (Ichimaru Pharcos Co., Ltd.)    -   Cucumber extract (Ichimaru Pharcos Co., Ltd.)    -   Kiwi extract (Ichimaru Pharcos Co., Ltd.)    -   Jujube extract ((Ichimaru Pharcos Co., Ltd.)    -   Tormentilla (Ichimaru Pharcos Co., Ltd.)

(a) Measurement of Cysteine Protease Enzyme Activity

80 μl of assay buffer (50 mM HEPES (pH 7.5)), 5 mM DTT, 2.5 mM EDTA and0.1% CHAPS) and 20 μl of 1 μg/ml papain (Sigma) were mixed and incubatedfor 15 minutes at room temperature. Next, 20 μl of 2.5 mMNα-benzoyl-L-arginine 4-nitroanilide hydrochloride (L-BAPNA) substratewere added followed by incubating for 15 minutes at 37° C. 30 μl of a25% acetic acid solution in ethanol and 30 μl of 0.2%p-dimethylaminocinnamaldehyde were added thereto to develop colorfollowed by measurement of absorbance at 545 nm. This absorbance wasdefined as the enzyme activity [x] of cysteine protease.

(b) Measurement of Enzyme Activity of System Containing

Candidate Herbal Medicine for Inhibiting Parakeratosis and CysteineProtease

60 μl of the aforementioned assay buffer and 20 μl of candidatesubstance were incubated for 30 minutes at room temperature. 20 μl of 1μg/ml papain were added thereto followed by incubating for 15 minutes atroom temperature. Next, 20 μl of 2.5 nM L-BAPNA were added followed byincubating for 15 minutes at 37° C. 30 μl of a 25% acetic acid solutionin ethanol and 30 μl of 0.2% p-dimethylaminocinnamaldehyde were added todevelop color followed by measurement of absorbance at 545 nm. Thisabsorbance was defined as the enzyme activity of cysteine protease [y],the percentage (%) of each tested compound with respect to theaforementioned [x] is shown in the column entitled “Sample only” in thefollowing Table 2.

The values shown in the “Sample only” column serve as indicators of thecysteine protease inhibitory activity of the tested herbal medicines perse, or in other words, the closer that value is to 100, the lower thecysteine protease inhibitory activity of the test herbal medicine. Inaddition, the values of [100−[sample only]], obtained by subtracting thevalue of “sample only” from 100, are also shown in Table 2. In thiscase, the closer that value is to zero, the lower the cysteine proteaseinhibitory activity of the tested herbal medicine.

(c) Measurement of Enzyme Activity of System Containing

SCCA-1, Candidate Herbal Medicine and Cysteine Protease

40 μl of the aforementioned assay buffer and 20 μl of recombinant SCCA-1were mixed followed by the addition of 20 μl of a candidate herbalmedicine and incubating for 30 minutes at room temperature. 20 μl of 1μg/ml papain were then added thereto followed by incubating for 15minutes at room temperature. Next, 20 μl of 2.5 mM L-BAPNA were addedfollowed by incubating for 15 minutes at 37° C. 30 μl of 25% acetic acidsolution in ethanol and 30 μl of 0.2% p-dimethylamino-cinnamaldehydewere added to develop color followed by measurement of absorbance at 545nm. This absorbance was defined as the enzyme activity [z] of cysteineprotease, and percentage (%) of each tested substance with respect tothe aforementioned [x] is shown in the column entitled “SCCA-1+” in thefollowing Table 2.

The value of “SCCA-1+” is an indicator of the total cysteine proteaseinhibitory activity of SCCA-1 and the cysteine protease inhibitoryactivity of the tested herbal medicine per se, or in other words, thecloser that value is to 100, the lower the total inhibitory activitythereof.

In addition, the column entitled “Difference” in the table indicates thevalue obtained by subtracting the value of “Sample only” from the valueof “SCCA-1+”. A large difference indicates that the cysteine proteaseinhibitory activity of SCCA-1 in the system containing SCCA-1, candidateherbal medicine and cysteine protease is low. In other words, thissuggests that suppression of the cysteine protease inhibitory activityof SCCA-1 by the candidate herbal medicine is remarkable.

The results are summarized in Table 2 below.

TABLE 2 [Sample 100 − [Sample Extract [SCCA-1+] only] only] [Difference]Kiwi 16.6 88.0 12.0 4.6 Cucumber 12.2 92.7 7.3 5.0 Jujube 14.2 88.8 11.23.1 Tomato 13.3 92.7 7.3 6.0 Cattail 49.8 66.5 33.5 16.3 Grape 24.9 83.916.1 8.8 Tormentilla 10.3 2.3 97.7 −87.3

As a result, cattail extract, grape extract, tomato extract, cucumberextract, kiwi extract and jujube extract, and particularly cattailextract, were found to significantly suppress the cysteine proteaseinhibitory activity of SCCA-1. Thus, these extracts are expected to beeffective in inhibiting epidermal parakeratosis.

INDUSTRIAL APPLICABILITY

According to the present invention, a method and pharmaceuticalcomposition are provided for treating and/or preventing a diseaseselected from the group consisting of psoriasis and squamous cellcarcinoma. In addition, according to the present invention, a means forinhibiting and treating epidermal parakeratosis can be provided thatuses a completely novel approach differing from the prior art.

1. A method for inhibiting epidermal parakeratosis by inhibitingcaspase-14 inhibitory activity of SCCA-1 in epidermal cells to normalizeepidermal cell keratization.
 2. The method according to claim 1, whereincaspase-14 inhibitory activity of SCCA-1 in epidermal cells is inhibitedby applying to the skin a skin composition for external use containingas an active ingredient thereof one or a plurality of types of herbalmedicines selected from the group consisting of cattail extract, grapeextract, tomato extract, cucumber extract, kiwi extract and jujubeextract.
 3. The method according to claim 2, wherein the skincomposition for external use contains cattail extract.